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DNA replication Euk

DNA replication Eukaryotes

Many origin sites in Eukaryotes, The initiations of DNA synthesis requires formation of initiation complex that contains a number of proteins.

Process after recruitment of an origin replication complex

Minichromosonme maintenance proteins are loaded onto the DNA by Cdc6 and Cdt1 to form a prereplication complex.

MCM complex

A hexamer of 6 MCM proteins and functions as a helicase

Prereplication complex

Formed in the nucleus before S phase. At the start of S phase Cdc6, Cdt1 are removed by inactivation or degradation. Once activated and DNA synthesis begins, no new MCMs can be loaded at the origin. (Licensing)

Regulation of DNA synthesis at origin

Regulated in a process called origin firing by S-phase cyclin/cdks. Recruits Pol alpha, which synthesizes the RNA/DNA primer. RFC loads PCNA at primer end

What replaces the RFC after it loads PCNA?

Pol-delata/pol epsilon. This assembles the leading strand synthesis. Pol alpha then makes primers on the strands on which the lagging strand synthesis takes place

What does the primase activity synthesize?

A short stretch of RNA which is extended by Pol alpha to add a short stretch of DNA of ca 20.

Does Pol alpha contain a proofreading activity?

Not very processive, does not contain a proofreading activity. Thus the primer for DNA synthesis in eukaryotes is a RNA-DNA molecule

Pol-delta

Multi-subunit enzyme, and the catalytic subunit has both a polymerase and a 3' to 5' exonuclease site. Functions at lagging strand. Interacts with PCNA which is the eukaryotic sliding clamp

Pol-epsilon

Has a polymerase and a 3' to 5' exonuclease site and functions at the leading strand.

PCNA (proliferating cell nuclear antigen)

Donut shaped. Endows Pol delta with a high degree of processivity.

What happens when pol delta encounters end of okazaki fragment?

Its progress is blocked, it is able to continue synthesis and displaces a short stretch of the 5' end of the primer to create a "FLAP" of several nucleotides. The flap is cleaved by flap endonuclease 1.

Endonuclease 1

Specific type of endonuclease which recognizes flap structures and cleaves the flap at the juncture to leave a nick. The process is continued until primer is removed and nick is sealed by DNA ligase.

Limitations to Flap endonuclease

It will not remove flaps longer than 10nts. If a longer flap is formed then endonuclease , DNA2 cleaves it.

RFC

RFC is the clamp loader. It is a multi-subunit protein. It recognizes the primer terminus, opens the PCNA ring and loads it onto DNA in an ATP dependent reaction and then displaced by Pol delta.

GINS complex and Cdc45

GINS complex encircles the leading strand template and interacts with pol e. They are held together with a bridge protein , Cdc45. Cdc45 is the key as it bings pol delta and epsilon.

Human chromosomes

Linear and the free ends of the chromosomes have special sequences that protect the ends.

Telomeric sequences

Several kb long G-T rich repeats of 6-8 nts sequence. At the very end there is a single stranded overhang that is thought to loop back and insert into the telomere and is stabilized by telemore proteins.

Problems that telemeric replications cause

Since polymerases act only in the 5' to 3' directions, the lagging strand would be incomplete as it requires a primer. Telomerase performs replication.

Telomerase

It is a reverse transcriptase, that synthesizes DNA onto an RNA primer. It carries its own RNA primer that is complementary to the telomere repeat sequence. Bind to 3' end and extend using RNA template.

AZT facts

A nucleoside/antiviral analog for HIV. It is classified as a NRTI=nucleoside reverse transcriptase inhibitor. It is taken up by cells and converted to AZT triphosphate by cellular kinases.

AZT as a substrate analog

Acts as a substrate analog of dTTP for the enzyme reverse transcriptase, which catalyzes incorporation of AZT-TP into viral DNA in place of dTTP, resulting in premature termination of viral DNA synthesis.